Function and Regulation of the ETS Transcriptional Repressor Tel-1/YAN

描述（申请人提供）：本提案是对1 R 01 GM 080372 -01“ETS转录抑制因子TEL 1/Yan的功能和调控”延续申请的修订。发育程序由转录因子驱动，这些转录因子作用于顺式调节增强子元件以协调基因表达的精确模式。转录因子保守ETS家族的两个成员，阻遏物Yan和激活物Pointed，在人类中被称为TEL 1和ETS 1/ETS 2，作为受体酪氨酸激酶/Ras/MAPK信号通路的下游效应子，协调各种细胞类型中增殖和分化之间的平衡。除了ETS DNA结合基序之外，Yan/TEL 1和Pointed/ETS 1/2共享第二个保守结构域，不育α基序（SAM），其介导同型和异型蛋白质-蛋白质相互作用。SAM介导的相互作用在正常细胞命运转变过程中调节网络输出，并在靶向Tel 1基因座的染色体易位产生的功能融合中提供致癌驱动力。该建议的目标是阐明Yan/TEL 1介导的抑制机制，并了解同型SAM介导的相互作用对这些机制的贡献。因为我们正在研究的信号分子和网络在哺乳动物中具有保守的功能，并且因为它们的失调有助于许多白血病和实体瘤的致癌转化，这些研究的发现将提高我们对人类发育和疾病机制的理解。目的1研究阻遏蛋白Yan被募集到染色质中调控基因表达的分子机制。我们将定义Yan-Pointed竞争如何在染色质占据和靶基因表达调控水平上表现出来。我们将测试的挑衅性的想法，除了其典型的作用作为一个序列特异性DNA结合阻遏物，严也作为一个染色质相关因子。目的2将探讨这一假设，严的复杂的染色质占有概况反映了一种新的机制，缓冲基因表达对遗传和环境噪音。使用最先进的体内分子遗传学技术，我们将确定Yan结合区域在最佳条件下以及面对遗传或环境变异时对调节其靶基因表达的个体贡献。使用3C和ChIP-qPCR，我们将研究三维染色质相互作用对Yan占用和基因表达调控的影响。目的3研究Yan主动抑制的分子机制。使用分子遗传学，生物信息学和生物化学方法的综合组合，我们将确定Yan转录复合物的组成部分，确定Yan单体与聚合物的差异阻遏活性，并探索聚合的程度如何影响体内的阻遏活性。

DESCRIPTION (provided by applicant): This proposal is a revision of renewal application for 1R01GM080372-01 "Function and regulation of the ETS transcriptional repressor TEL1/Yan". Developmental programs are driven by transcription factors that act upon cis-regulatory enhancer elements to coordinate precise patterns of gene expression. Two members of the conserved ETS family of transcription factors, a repressor Yan and an activator Pointed, known as TEL1 and ETS1/ETS2 in humans, act as downstream effectors of the receptor tyrosine kinase/Ras/MAPK signaling pathway to orchestrate the balance between proliferation and differentiation in a variety of cell types. In addition to the ETS DNA binding motif, Yan/TEL1 and Pointed/ETS1/2 share a second conserved domain, the sterile alpha motif (SAM), which mediates homo- and hetero-typic protein-protein interactions. SAM-mediated interactions modulate network output during normal cell fate transitions and provide an oncogenic driving force in the functional fusions produced by chromosomal translocations targeting the Tel1 locus. The goals of this proposal are to elucidate the mechanisms of Yan/TEL1-mediated repression and to understand the contribution of homotypic SAM- mediated interactions to these mechanisms. Because the signaling molecules and networks we are studying have conserved functions in mammals, and because their dysregulation contributes to oncogenic transformation in a number of leukemia and solid tumors, the discoveries resulting from these investigations will improve our understanding of mechanisms underlying human development and disease. Aim 1 will investigate the molecular mechanisms by which the repressor Yan is recruited to chromatin to regulate gene expression. We will define how Yan-Pointed competition manifests at the level of chromatin occupancy and regulation of target gene expression. We will test the provocative idea that in addition to its canonical role as a sequence-specific DNA binding repressor, Yan also acts as a chromatin associated factor. Aim 2 will explore the hypothesis that Yan's complex chromatin occupancy profile reflects a novel mechanism for buffering gene expression against genetic and environmental noise. Using state of the art in vivo molecular genetics technologies, we will determine the individual contribution of Yan-bound regions to regulating the expression of its target genes under both optimal conditions and in the face of genetic or environmental variation. Using 3C and ChIP-qPCR, we will investigate the influence of three-dimensional chromatin interactions on Yan occupancy and regulation of gene expression. Aim 3 will investigate the molecular mechanisms of active Yan-mediated repression. Using an integrative combination of molecular genetics, bioinformatics and biochemical approaches, we will identify the components of Yan transcriptional complexes that determine the differential repressor activity of Yan monomers versus polymers and explore how the extent of polymerization impacts repressive activity in vivo.